PROJECT SUMMARY/ABSTRACT T cell immunotherapy is emerging as a promising cancer treatment option and has proven effective in a range of malignancy. However, a concern has been that prolong circulation and/or non-specific migration of the adoptively transferred in vitro activated T cells to non-target tissue sites might predispose to cardiovascular damages and systemic inflammatory responses. Anecdotal evidence of a cardiovascular hazard has emerged and abundant data point to exacerbation of cytokine release syndrome associated with T cell immunotherapy. We undertook this study to address critical knowledge gaps regarding the molecular mechanisms that determine the function and fate of the adoptively transferred in vitro-generated T cells, and cardiovascular toxicity associated with sequestration of the therapeutic T cells at non-tumor-bearing tissues after intravenous transfer. Through several lines of evidence from our preliminary study, we propose that autologous T cells undergo significant molecular and cellular reprogramming during ex-vivo manufacturing process. We predict that the intrinsic changes are important for the robust T cell activation and expansion, but fail to derive T cell migration toward the target tumor, and thus serve to increase toxicity. We discovered that a decrease in ?II-spectrin expression during in vitro T cell activation results in decreased cell stiffness and a dramatic change in spontaneous T cell migration pattern upon intravenous transfer. Moreover, screening of a key intracellular protein associated with the altered T cell migration revealed a novel Rab13-mediated endosomal redistribution pattern that mediates the non-specific T cell migration. We will, (1) determine the causes of cardiovascular cytotoxicity and cytokine release syndrome associated with non-specific migration of in vitro activated T cells, (2) determine the molecular mechanisms that prevent specific migration toward the target tissue site, and (3) test whether we can generate T cells with an improved tissue-specific homing property and a reduced cardiovascular side-effects. These studies will combine differential perturbations of novel mechanisms that regulate activated T cell migration, in vivo mouse models, state of the art intravital multiphoton imaging, high-resolution singles cell assays, and analysis defining vascular inflammatory responses to understand a potentially serious risk of adoptively T cell transfer immunotherapy. We shall also explore novel alternative approaches that might promote the anti-cancer efficacy and minimize the cardiovascular risk of the T cell immunotherapy.